基于结构类似物的交叉反应性制备高灵敏度 玉米赤霉醇单克隆抗体

doi:10.3864/j.issn.0578-1752.2020.05.017

摘要/Abstract

摘要：

【目的】获取高灵敏度的玉米赤霉醇（zearalanol,ZAL）单克隆抗体,为提高玉米赤霉醇免疫学检测方法灵敏度研究奠定基础。【方法】利用ZAL的结构类似物玉米赤霉酮（zearalanone, ZAN）制备人工完全抗原。肟化改造ZAN得到ZAN-O;碳二亚胺（EDC）法把ZAN-O分别连接到牛血清白蛋白（BSA）和卵清蛋白（OVA）上制备出ZAN-O-BSA和ZAN-O-OVA。ZAN-O-BSA免疫小鼠,免疫剂量为50 μg蛋白/只鼠。选取血清效价高、灵敏度好的小鼠进行细胞融合。阳性杂交瘤筛选过程中,利用ZAL替代ZAN作为阻断剂,筛选能分泌抗ZAL单克隆抗体的杂交瘤细胞。体内诱生腹水法来批量制备ZAL单抗,并对单抗的免疫学性能进行了鉴定。【结果】通过细胞融合,阳性杂交瘤筛选得到了1株能分泌抗ZAL单克隆抗体的杂交瘤细胞,命名为12B10A7,其分泌的ZAL单抗灵敏度(半数抑制浓度, IC₅₀)为577 pg?mL ^-1,亲和力常数Ka=6.21×10 ⁷L?mol ^-1,与结构类似物β-玉米赤霉醇（β-zearalanol, β-ZAL）、α-玉米赤霉烯醇（α-zearalenol, α-ZEL）、β-玉米赤霉烯醇（β-zearalenol, β-ZEL）、玉米赤霉酮、玉米赤霉烯酮（zearalenone, ZEN）分别有43.06%、15.51%、15.22%、77.65%及9.79%的交叉反应率,而与其他霉菌毒素及载体蛋白交叉反应率均<0.06%。【结论】基于抗体交叉反应特性,利用ZAN制备人工抗原,得到了ZAL的单克隆抗体,所制备的单克隆抗体亲和力高,灵敏度好,特异性强。

关键词: 玉米赤霉醇, 玉米赤霉酮, 结构类似物, 交叉反应, 单克隆抗体

Abstract:

【Objective】The aim of this study was to obtain highly sensitive monoclonal antibody of Zearalanol (ZAL). 【Method】Zearalanone (ZAN), as the structural analogs of ZAL, was employed to synthesize the artificial complete antigen. ZAN structure was modified by oximation reaction to obtain ZNA-O. ZAN-O was coupled with bovine serum albumin and ovalbumin by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) respectively to prepare ZAN-O-BSA and ZAN-O-OVA. ZAN-O-BSA was used to immunize the mice with the immune dose of 50 μg protein/mouse. The mouse with higher serum titer and sensitivity was selected for cell fusion. During the process of cell fusion to screen the positive hybridoma cells, ZAL was used as a blocker instead of ZAN to screen the hybridoma cell lines that could secrete monoclonal antibody against ZAL. ZAL monoclonal antibody was prepared in batches by ascites induction in vivo, and the immunological properties of the monoclonal antibodies were identified. 【Result】By cell fusion and screening for positive hybridoma cell lines, one hybridoma cell line named 12B10A7 that could secrete monoclonal antibody against ZAL was obtained. The sensitivity (half inhibitory concentration, IC₅₀) of the prepared monoclonal antibody was 577 pg?mL ^-1, the affinity constant Ka was 6.21×10 ⁷ L?mol ^-1, the cross-reactivity rate with structural analogues such as β-zearalanol, α-zearalenol, β-zearalenol, zearalanone and zearalenone were 43.06%, 15.51%, 15.22%, 77.65% and 9.79%, respectively, and the cross reaction rates with other toxins and carrier proteins were all less than 0.06%. 【Conclusion】In conclusion, based on the cross reaction characteristics of antibody, in this study, we prepared the artificial antigen of ZAN, and got the monoclonal antibody against ZAL, which had high affinity, sensitivity and specificity.

Key words: Zearalanol, Zearalanone, structural analogs, cross reaction, monoclonal antibody

胡骁飞,李青梅,姚静静,胡思宇,孙亚宁,邢云瑞,邓瑞广,张改平. 基于结构类似物的交叉反应性制备高灵敏度玉米赤霉醇单克隆抗体[J]. 中国农业科学, 2020, 53(5): 1071-1080.

HU XiaoFei,LI QingMei,YAO JingJing,HU SiYu,SUN YaNing,XING YunRui,DENG RuiGuang,ZHANG GaiPing. Development of High Sensitive Zeranol Monoclonal Antibody Based on the Cross Reactivity of Structural Analogs[J]. Scientia Agricultura Sinica, 2020, 53(5): 1071-1080.

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ZEN mAb稀释倍数 Dilution ratio of ZEN mAb	1×10⁴	2×10⁴	4×10⁴	8×10⁴	16×10⁴	32×10⁴	64×10⁴	空白 Blank
ZAN-O-BSA	>3.5	3.044	1.847	1.028	0.579	0.343	0.243	0.064
ZAN-O-OVA	1.182	0.869	0.653	0.387	0.289	0.196	0.163	0.042

小鼠编号 Number of mice	血清稀释倍数 Dilution ratio of serum
小鼠编号 Number of mice	200	400	800	1600	3200	6400	12800	空白Blank
1	3.471	2.438	1.462	0.718	0.418	0.329	0.240	0.105
2	2.656	1.898	1.047	0.611	0.345	0.244	0.205	0.083
3	>3.50	2.937	2.084	1.120	0.656	0.384	0.309	0.107
4	2.179	2.012	1.828	1.432	1.043	0.822	0.670	0.089
5	1.879	1.855	1.392	0.938	0.692	0.539	0.373	0.105
6	2.977	2.728	2.122	1.560	1.049	0.692	0.502	0.089

小鼠编号 Number of mice	ZAL浓度 ZAL concentration (ng·mL^-1)						阳性 Positive	空白 Blank
小鼠编号 Number of mice	500	250	125	62.5	31.25	15.62	阳性 Positive	空白 Blank
1	0.681	0.836	1.099	1.148	1.286	1.802	1.910	0.104
2	0.736	0.844	0.915	0.986	1.085	1.128	1.550	0.109
3	1.078	1.043	1.101	1.176	0.993	1.168	1.544	0.092
4	0.569	0.608	0.731	0.820	0.987	1.069	1.591	0.107
5	0.865	0.879	0.927	0.972	0.994	1.077	1.346	0.103
6	0.288	0.402	0.510	0.621	0.653	0.741	1.276	0.106